The aminoacyl-tRNA synthetases are responsible for pairing amino acids and tRNAs, and are crucial both for ribosomal protein synthesis and other processes outside translation. The aminoacyl-tRNA synthetase family also contains numerous paralogs that recapitulate either the catalytic core or the various appended domains of aminoacyl-tRNA synthetases. In bacteria the canonical lysyl-tRNA synthetase provides substrates both to translate lysine codons and to modify membrane lipids, while the closely related paralog PoxA instead participates in the post-translational modification of elongation factor P, a pathway critical for the virulence of pathogens such as Salmonella. In both instances, the ability of lysyl-tRNA synthetase-type proteins to promote lysylation of molecules other than tRNA has widespread effects on the cell, determining key properties such as antibiotic resistance. The objective of this proposal is to build on our earlier work on the lysyl-tRNA synthetase family to study non-canonical roles of lysyl-tRNA synthetases and their paralogs. Specifically, we will determine: i) The structural and functional basis of lysyl-tRNA's role in lipd modification. ii) The molecular basis of divergent substrate recognition by lysyl-tRNA synthetase and PoxA. iii) The mechanism of action of modified elongation factor P. This work will reveal the molecular mechanisms that allow members of the lysyl-tRNA synthetase family to participate in aminoacylation reactions outside protein synthesis, and will show how the aminoacylation of proteins and lipids can provide these molecules with new and essential biological activities. Understanding the structural and functional features that allow the lysyl-tRNA synthetase family to play such broad roles in the cell will also provide insights into possible new functions in othe aminoacyl-tRNA synthetase protein families, the vast majority of which contain numerous well-conserved paralogs with no known function. PUBLIC HEALTH RELEVANCE: Anthrax, Salmonellosis and Tuberculosis are infectious diseases with potentially fatal sequelae. Lysyl-tRNA synthetase family proteins contribute to the virulence of the causative agents of these and other diseases by modulating antibiotic resistance and other traits through mechanisms which to date have only been minimally defined. The proposed studies will define the roles of lysyl-tRNA synthetase and its paralog PoxA in establishing antibiotic resistance, and help determine how the corresponding pathways can be used as targets for the development of new anti-infective agents for the treatment of bacterial infectious diseases.